Transcription of pre-mRNA by RNA polymerase II (pol II) is very often the major regulatory step in differential gene expression in eukaryotic cells. Precise changes in the patterns of transcription are fundamental for normal cell growth and division, the proper response of a cell to its extracellular environment, and for the development and differentiation of multicellular organisms. Mis-regulation of gene expression can result in human disease, including cancer. It is therefore important to fully understand the basic components of the transcription machinery, their mechanism of action, and how their activities are regulated. Pol II-dependent transcription is an extremely complex process that requires the coordinated action of a large number of proteins, including pol II and other general transcription factors, numerous site-specific DNA-binding proteins, and chromatin-associated proteins. The long-term objective of this project is to understand the mechanisms that regulate pol II and the general transcription factors. To achieve this goal, we have utilized the powerful genetic, molecular, and biochemical techniques available in the budding yeast Saccharomyces cerevisiae. Because the proteins involved in transcription have been conserved throughout eukaryotes, these studies in a relatively simple organism are likely to provide valuable information into regulatory mechanisms that are also used in human cells. Two of the most commonly regulated components of the general transcription machinery are RNA polymerase II (pol II) and the TATA-binding protein (TBP). The binding of TBP to the TATA box is the first step in transcription, and it is thus frequently rate-limiting at many promoters. Pol II activity is highly regulated by phosphorylation of its largest subunit, which alters its association with accessory proteins and triggers the transition from initiation to elongation. The experiments described in this proposal focus on two genes that were identified in our lab as having important general roles in transcription: BURl encodes a cyclin-dependent protein kinase that phosphorylates the largest subunit of pol II, while BUR6 encodes a regulator of TBP. The experiments described in this proposal are designed to: 1) identify the role of the Bur1 kinase during the transcription cycle, 2) determine how Bur1 activity is regulated, and 3) investigate the positive effects of Bur6 on TBP activity.